Solomonseal Polysaccharide and Sulfated Codonopsis pilosula Polysaccharide Synergistically Resist Newcastle Disease Virus

Five combinations of three ratios (PS₉-sPS₁, PS₇-sPS₃ and PS₆-sPS₄) were prepared with polysaccharide (PS) and sulfated polysaccharide (sPS). The antiviral activities of these compounds were subsequently compared in vitro using the MTT assay, observation of the virus structure and immunofluorescence. The results demonstrated that SP₉-sCP₁, CP₇-sCA₃, EP₇-sAP₃, CA₉-sEP₁ and EP₇-sCA₃ presented higher activities, and SP₉-sCP₁ displayed the highest virus inhibition rate and clearly killed the virus and inhibited viral antigen expression. In an in vivo test, 28-day-old chickens were challenged with Newcastle disease virus (NDV) and were administered the five drug combinations. On day 14 after the challenge, the morbidity, mortality and cure rate in each group were calculated. The results indicated that SP₉-sCP₁ presented the lowest morbidity and mortality and the highest cure rate. These results indicate that Solomonseal polysaccharide and sulfated Codonopsis pilosula polysaccharide synergistically resist NDV. Moreover, SP₉-sCP₁ had the highest efficacy and may be used as a new antiviral drug.     

SEPTIN2 and STATHMIN Regulate CD99-Mediated Cellular Differentiation in Hodgkin's Lymphoma

Hodgkin’s lymphoma (HL) is a lymphoid neoplasm characterized by Hodgkin’s and Reed-Sternberg (H/RS) cells, which is regulated by CD99. We previously reported that CD99 downregulation led to the transformation of murine B lymphoma cells (A20) into cells with an H/RS phenotype, while CD99 upregulation induced differentiation of classical Hodgkin’s lymphoma (cHL) cells (L428) into terminal B-cells. However, the molecular mechanism remains unclear. In this study, using fluorescence two-dimensional differential in-gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), we have analyzed the alteration of protein expression following CD99 upregulation in L428 cells as well as downregulation of mouse CD99 antigen-like 2 (mCD99L2) in A20 cells. Bioinformatics analysis showed that SEPTIN2 and STATHMIN, which are cytoskeleton proteins, were significantly differentially expressed, and chosen for further validation and functional analysis. Differential expression of SEPTIN2 was found in both models and was inversely correlated with CD99 expression. STATHMIN was identified in the A20 cell line model and its expression was positively correlated with that of CD99. Importantly, silencing of SEPTIN2 with siRNA substantially altered the cellular cytoskeleton in L428 cells. The downregulation of STATHMIN by siRNA promoted the differentiation of H/RS cells toward terminal B-cells. These results suggest that SEPTIN2-mediated cytoskeletal rearrangement and STATHMIN-mediated differentiation may contribute to changes in cell morphology and differentiation of H/RS cells with CD99 upregulation in HL.     

Anaplastic Lymphoma Kinase Rearrangement in Digestive Tract Cancer: Implication for Targeted Therapy in Chinese Population

Background

Anaplastic lymphoma kinase (ALK) rearrangements define a subgroup of lung cancer which is eligible to targeted kinase inhibition. The aim of this study is to observe the incidence rate of ALK fusion in a large cohort of Chinese digestive tract cancer patients.

Patients and Methods

Tissue microarray (TMA) was constructed from 808 digestive tract cancer cases, including 169 esophageal squamous cell carcinoma, 182 gastric cancer and 457 colorectal cancer (CRC) cases. We tested all cases for ALK expression via a fully automated immunohistochemistry (IHC) assay. The IHC-positive cases were subjected to fluorescence in situ hybridization (FISH), real-time polymerase chain reaction (qRT-PCR), target gene enrichment and sequencing for confirmation of ALK gene rearrangement and discovery of novel fusion partner.

Results

Among the tested cases, 2 (0.44%) CRC cases showed positive both by IHC and FISH. By qRT-PCR, EML4–ALK fusion was found in one IHC-positive CRC case. In another IHC-positive CRC case, target gene enrichment and sequencing revealed ALK was fused to a novel partner, spectrin beta non-erythrocytic 1 (SPTBN1). One gastric cancer case showed partially positive IHC result, but no fusion was found by FISH and gene sequencing.

Conclusions

The incidence rate of ALK gene fusion in Chinese CRC patients was 0.44%,but not detectable in gastric and esophageal cancers. The novel SPTBN1 -ALK fusion, together with other ALK fusion genes, may become a potential target for anti-ALK therapy.     

Dynamic FDG-PET Imaging to Differentiate Malignancies from Inflammation in Subcutaneous and In Situ Mouse Model for Non-Small Cell Lung Carcinoma (NSCLC)

Background

[¹⁸F]fluoro-2-deoxy-D-glucose positron emission tomography (FDG-PET) has been widely used in oncologic procedures such as tumor diagnosis and staging. However, false-positive rates have been high, unacceptable and mainly caused by inflammatory lesions. Misinterpretations take place especially when non-subcutaneous inflammations appear at the tumor site, for instance in the lung. The aim of the current study is to evaluate the use of dynamic PET imaging procedure to differentiate in situ and subcutaneous non-small cell lung carcinoma (NSCLC) from inflammation, and estimate the kinetics of inflammations in various locations.

Methods

Dynamic FDG-PET was performed on 33 female mice inoculated with tumor and/or inflammation subcutaneously or inside the lung. Standardized Uptake Values (SUVs) from static imaging (SUVmax) as well as values of influx rate constant (Ki) of compartmental modeling from dynamic imaging were obtained. Static and kinetic data from different lesions (tumor and inflammations) or different locations (subcutaneous, in situ and spontaneous group) were compared.

Results

Values of SUVmax showed significant difference in subcutaneous tumor and inflammation (p<0.01), and in inflammations from different locations (p<0.005). However, SUVmax showed no statistical difference between in situ tumor and inflammation (p = 1.0) and among tumors from different locations (subcutaneous and in situ, p = 0.91). Values of Ki calculated from compartmental modeling showed significant difference between tumor and inflammation both subcutaneously (p<0.005) and orthotopically (p<0.01). Ki showed also location specific values for inflammations (subcutaneous, in situ and spontaneous, p<0.015). However, Ki of tumors from different locations (subcutaneous and in situ) showed no significant difference (p = 0.46).

Conclusion

In contrast to static PET based SUVmax, both subcutaneous and in situ inflammations and malignancies can be differentiated via dynamic FDG-PET based Ki. Moreover, Values of influx rate constant Ki from compartmental modeling can offer an assessment for inflammations at different locations of the body, which also implies further validation is necessary before the replacement of in situ inflammation with its subcutaneous counterpart in animal experiments.     

Cytokine and Antibody Based Diagnostic Algorithms for Sputum Culture-Positive Pulmonary Tuberculosis

Background

Tuberculosis (TB) is one of the most serious infectious diseases globally and has high mortality rates. A variety of diagnostic tests are available, yet none are wholly reliable. Serum cytokines, although significantly and frequently induced by different diseases and thus good biomarkers for disease diagnosis and prognosis, are not sufficiently disease-specific. TB-specific antibody detection, on the other hand, has been reported to be highly specific but not sufficiently sensitive. In this study, our aim was to improve the sensitivity and specificity of TB diagnosis by combining detection of TB-related cytokines and TB-specific antibodies in peripheral blood samples.

Methods

TB-related serum cytokines were screened using a human cytokine array. TB-related cytokines and TB-specific antibodies were detected in parallel with microarray technology. The diagnostic performance of the new protocol for active TB was systematically compared with other traditional methods.

Results

Here, we show that cytokines I-309, IL-8 and MIG are capable of distinguishing patients with active TB from healthy controls, patients with latent TB infection, and those with a range of other pulmonary diseases, and that these cytokines, and their presence alongside antibodies for TB-specific antigens Ag14-16kDa, Ag32kDa, Ag38kDa and Ag85B, are specific markers for active TB. The diagnostic protocol for active TB developed here, which combines the detection of three TB-related cytokines and TB-specific antibodies, is highly sensitive (91.03%), specific (90.77%) and accurate (90.87%).

Conclusions

Our results show that combining detection of TB-related cytokines and TB-specific antibodies significantly enhances diagnostic accuracy for active TB, providing greater accuracy than conventional diagnostic methods such as interferon gamma release assays (IGRAs), TB antibody Colloidal Gold Assays and microbiological culture, and suggest that this diagnostic protocol has potential for clinical application.     

Effect of P144? (Anti-TGF-β) in an “In Vivo” Human Hypertrophic Scar Model in Nude Mice

Background

Hypertrophic scars are one of the most important complications in surgery due to their cosmetic and functional impairments. Previous studies in tissue fibrotic disorders have shown promising results by inhibiting the biological activity effect of Transforming Growth Factor-beta 1 (TGF-β1). The aim of the current study was to determine the clinical effect of the inhibition of TGF-β1 signaling in human hypertrophic scars implanted in nude mice by topical application of an inhibitor of TGF-β1 (P144®).

Material and Methods

A total of 30 human hypertrophic scars were implanted in 60 nude mice. The animals were divided in two groups, group A (placebo) and group B (treatment). Group C (basal) was considered as the preimplanted scar samples and they were not implanted in the nude mice. After the shedding period, topical application of a lipogel containing placebo (group A) or P144 (group B) was daily administered during two weeks. The animals were sacrificed upon completion of the study. Total area, thickness and collagen fibers area were measure and compared across all groups. Immunohistochemistry was also performed in order to quantify collagen type I and type III and elastic fiber expressions present in the dermis.

Results

Successful shedding was achieved in 83,3% of the xenografts. The mean time for shedding was 35±5.4 days. Statistically significant differences were found in the total area, collagen fibers area and thickness between the groups. Increased elastic fibers and decreased collagen I were found in the P144-treated group compared to the basal group.

Conclusion

Topical application of an inhibitor of TGF-β1 may promote scar maturation and clinical improvement of hypertrophic scar morphology features in an “in vivo” model in nude mice after two weeks of treatment.     

Investigation on the Tribological Behavior and Wear Mechanism of Five Different Veneering Porcelains

Objectives

The primary aim of this research was to investigate the wear behavior and wear mechanism of five different veneering porcelains.

Methods

Five kinds of veneering porcelains were selected in this research. The surface microhardness of all the samples was measured with a microhardness tester. Wear tests were performed on a ball-on-flat PLINT fretting wear machine, with lubrication of artificial saliva at 37°C. The friction coefficients were recorded by the testing system. The microstructure features, wear volume, and damage morphologies were recorded and analyzed with a confocal laser scanning microscope and a scanning electron microscope. The wear mechanism was then elucidated.

Results

The friction coefficients of the five veneering porcelains differ significantly. No significant correlation between hardness and wear volume was found for these veneering porcelains. Under lubrication of artificial saliva, the porcelain with higher leucite crystal content exhibited greater wear resistance. Additionally, leucite crystal size and distribution in glass matrix influenced wear behavior. The wear mechanisms for these porcelains were similar: abrasive wear dominates the early stage, whereas delamination was the main damage mode at the later stage. Furthermore, delamination was more prominent for porcelains with larger crystal sizes.

Significance

Wear compatibility between porcelain and natural teeth is important for dental restorative materials. Investigation on crystal content, size, and distribution in glass matrix can provide insight for the selection of dental porcelains in clinical settings.     

CD40 ligand induces RIP1-dependent,necroptosis-like cell death in low-grade serous but not serous borderline ovarian tumor cells

Ovarian high-grade serous carcinomas (HGSCs) and invasive low-grade serous carcinomas (LGSCs) are considered to be distinct entities. In particular, LGSCs are thought to arise from non-invasive serous borderline ovarian tumors (SBOTs) and show poor responsiveness to conventional chemotherapy. The pro-apoptotic effects of CD40 ligand (CD40L) have been demonstrated in HGSC, though the underlying mechanisms are not fully understood. Conversely, the therapeutic potential of the CD40L-CD40 system has yet to be evaluated in LGSC. We now show that CD40 protein is focally expressed on tumor cells in two of five primary LGSCs compared with no expression in eight primary SBOTs. Treatment with CD40L or agonistic CD40 antibody decreased the viability of LGSC-derived MPSC1 and VOA1312 cells, but not SBOT3.1 cells. Small interfering RNA (siRNA) targeting CD40 was used to show that it is required for these reductions in cell viability. CD40L treatment increased cleaved caspase-3 levels in MPSC1 cells though, surprisingly, neither pan-caspase inhibitor nor caspase-3 siRNA reversed or even attenuated CD40L-induced cell death. In addition, CD40-induced cell death was not affected by knockdown of the mitochondrial proteins apoptosis-inducing factor (AIF) and endonuclease G (EndoG). Interestingly, CD40L-induced cell death was blocked by necrostatin-1, an inhibitor of receptor-interacting protein 1 (RIP1), and attenuated by inhibitors of RIP3 (GSK''872) or MLKL (mixed lineage kinase domain-like; necrosulfonamide). Our results indicate that the upregulation of CD40 may be relatively common in LGSC and that CD40 activation induces RIP1-dependent, necroptosis-like cell death in LGSC cells.Epithelial ovarian cancer accounts for approximately 90% of all ovarian malignancies and is the leading cause of gynecological cancer death in developed countries.^{1, 2} Recently, differences in molecular alterations and clinicopathological features have established a dualistic model dividing ovarian serous carcinomas into high-grade serous carcinoma (HGSC) and low-grade serous carcinoma (LGSC) subtypes. HGSCs are more common and are thought to develop directly from the ovarian surface epithelium or from serous tubal intra-epithelial carcinomas in the fallopian tube. In contrast, LGSCs are rare and are generally considered to develop from benign serous cystadenomas through serous borderline ovarian tumors (SBOT). SBOTs are slow-growing, non-invasive epithelial neoplasms that have a better prognosis compared with other types of ovarian cancer.^{3, 4, 5} Our previous studies have shown that the inhibition of p53 or treatment of epidermal growth factor or transforming growth factor-β1 increases SBOT cell invasion by inducing epithelial–mesenchymal transition, which suggests a possible mechanism that mediates the progression from SBOT to LGSC.^{6, 7, 8, 9} However, many of SBOTs recur as LGSCs that display poor responsiveness to conventional chemotherapy and for which survival rates are <50%.^{1, 3, 10} Thus, the development of novel, targeted therapeutic strategies is likely required to significantly improve patient survival.CD40, a transmembrane glycoprotein belonging to the tumor necrosis factor receptor superfamily, is expressed by a wide range of cell types including immune, endothelial and epithelial cells. Engagement of CD40 with its ligand, CD40L, has been shown to have important roles in a variety of physiological and pathological processes, especially in immunity.^{11, 12} In addition, CD40 expression has been demonstrated in several types of cancer, including colon, lung, cervical, bladder and prostate cancer.¹³ However, reported functions of CD40 in tumor cells vary, with both pro-apoptotic and anti-proliferative effects observed depending on the cellular context.^{14, 15, 16} Alternatively, some studies have shown that CD40 activation may promote the neoplastic transformation and growth of normal cells.^{17, 18, 19} Expression of CD40 has been demonstrated in ovarian cancer cell lines and tumor samples, but not in normal ovarian tissue, suggesting that CD40 may have an important role in ovarian tumors.^{20, 21, 22, 23, 24} Indeed, CD40L-CD40 signaling has been shown to induce growth-inhibitory effects in HGSC cells,^{20, 21, 23, 24, 25} however, the therapeutic potential of CD40 in LGSC and SBOT has not been evaluated.In the present study, we report for the first time elevated CD40 expression in a significant proportion of LGSCs compared with SBOTs. Moreover, CD40 expression is elevated in LGSC-derived MPSC1 and VOA1312 cells compared with SBOT3.1 cells, and CD40 activation induces cell death via CD40 only in LGSC-derived cells. Neither pan-caspase inhibitor nor caspase-3 small interfering RNA (siRNA) has any effect on CD40L-induced MPSC1 cell death. Moreover, CD40L-induced cell death was unaffected by individual or combined knockdown of the mitochondrial proteins apoptosis-inducing factor (AIF) and endonuclease G (EndoG). Interestingly, our results suggest that receptor-interacting protein 1 (RIP1), RIP3 and MLKL are involved in CD40-induced MPSC1 cell death. These results demonstrate that CD40 induces RIP1-dependent, necroptosis-like cell death in LGSC cells.